This invention is related to fused pyrazine derivatives and adhesion molecules expression inhibitors containing fused pyrazine derivatives as active ingredient. More particularly, this invention is related to adhesion molecules expression inhibitors containing fused pyrazine derivatives of the formula (I): 
wherein all the symbols are the same meaning as hereinafter defined, and non-toxic salts thereof, and novel fused pyrazine derivatives of the above formula (I) and non-toxic salts thereof, and processes for the preparation thereof.
Cellular adhesions are basic responses in the organism and relate to various biological phenomenon deeply. In diseases, there are various examples which are mediated by excess reactions or disordered reactions. Inflammatory reaction, which protect a host from a foreign matter, is essentially one of the defensive response in the organism and it is thought that acceleration of leukocytes adhesion to endothelial cells is a central process in the early step in this reaction. Leukocytes are one of the major cells in inflammation and migrate into the inflammatory tissues and secrete chemical mediators, cytokines or enzymes and develop inflammation. Therefore, it has been thought leukocytes extravasation from the vascular flow is an important process in inflammatory development and the process, which is leukocytes-endothelial cells adhesion, is essential in the early step in transmigration.
Cellular migration is at least classified following 4 steps:
1) tethering of leukocytes to endothelial cells,
2) rolling of leukocytes,
3) firm adhesion of leukocytes to endothelial cells,
4) transmigration of leukocytes.
Recently, it has been reported these various cellular adhesions are mediated by cell surface molecular groups which are called adhesion molecules and explained the distinct molecular groups specifically play a central role in the previous each adhesion step. That is to say, it has been explained tethering and rolling process are induced by interaction of carbohydrate and selectin, such as E-selectin, and subsequent firm adhesion and extravasation processes are mediated by interaction of integrin family on leukocytes and immunoglobulin superfamily, such as ICAM-1 (Intercellular Adhesion Molecule-1) and VCAM-1 (Vascular Cell Adhesion Molecule-1).
Any of endothelial adhesion molecules which are E-selectin, VCAM-1 and ICAM-1 are induced molecules stimulated by inflammatory cytokines, such as TNF and IL-1. In fact, it has been reported that these adhesion molecules expression are upregulated in the various lesional sites. Therefore the upregulation of adhesion molecules develops cellular adhesion and participates in disease formation, such as chronic inflammation.
It has been suggested these cellular adhesions to endothelial cells participate not only inflammatory reaction but also tumor metastasis, allergic reaction and immune reaction. Furthermore, the reports which showed the upregulation of VCAM-1 and the increase of ICAM-1 concentration in HIV infected patients have suggested the relation between adhesion molecules expression and HIV infection [Clinical Immunology and Immunopathology, 81, 6-21 (1996)].
From these viewpoints, it is expected the inhibition of adhesion molecules expression which are E-selectin, VCAM-1 and ICAM-1 suppresses the cellular adhesion and links the treatment for various diseases.
The present invitation provides a useful new therapeutic agent which has inhibitory activity on these adhesion molecules expression
According to a further aspect, the present invitation provides the use of the compounds for treatment and/or prevention of disorders mediated by cellular adhesion and infiltration, such as inflammation, rheumatoid arthritis, allergies, asthma, atopic dermatitis, psoriasis, suppression of ischemia reperfusion injury, nephritis, hepatitis, multiple sclerosis, ulcerative colitis, adult respiratory distress syndrome (ARDS), suppression of transplant rejection, sepsis, diabetes, autoimmune diseases, tumor metastasis, arteriosclerosis and AIDS [The Hand Book of Immunopharmacology, Adhesion Molecule, Academic Press, (1994), Trends in Pharmacological Science 16, 418-423, (1995), Molecular Medicine Today, 3, 418-423 (1997), Molecular Medicine Today, 3, 310-321 (1997), Japanese Journal of Inflammation, 17, 459-467 (1997)].
For example, 1,2,4-triazolo-[4,3-a]pyrazine derivatives of the formula (X): 
in the specification of GB 1235910,
and the formula (Y): 
in the specification of GB 1146770, are disclosed to be useful as treatment of bronchial disorder.
In the specification of U.S. Pat. Nos. 4,200,750, 4,198,508, 4,191,767, 4,191,766, BE 878028 and BE 862608, imidazo-[1,2-a]quinoxaline derivatives are disclosed to be useful as immunosuppressants, anti-inflammatory agents, antifungal agents, antiyeast agents, treatment of bronchial disorder.
Besides, in the specification of WO 9535296, fused imidazole derivatives of the formula (Z): 
are disclosed to have an inhibitory activity of adhesion molecules expression.
Energetic investigations have been carried out in order to make adhesion molecules expression inhibitors. The present inventors have found that fused pyrazine derivatives of the formula (I) accomplished the present purpose.
Fused pyrazine derivatives of the formula (I) of the present invention are not known as adhesion molecules expression inhibitors at all. Besides, a lot of fused pyrazine derivatives of the formula (I) are novel compounds.
The present invention is related to:
(i) adhesion molecules expression inhibitors containing fused pyrazine derivatives of the formula (I): 
wherein R1 and R2 each, independently, is (i) hydrogen, (ii) C1-8 alkyl, (iii) C1-8 alkoxy, (iv) C1-8 alkylthio, (v) Cyc1, (vi) nitrile, (vii) formyl, (viii) xe2x80x94COOR14, in which R14 is hydrogen or C1-8 alkyl, (ix) xe2x80x94CONR15R16, in which R15 and R16 each, independently, is hydrogen, C1-8 alkyl or phenyl, (x) C1-8 alkyl or C2-8 alkenyl substituted by 1 or 2 of hydroxy, C1-4 alkoxy, phenoxy, halogen atom, nitrile, C2-5 acyl, xe2x80x94COOR14, xe2x80x94CONR15R16, or xe2x80x94NR17R18, in which R17 and R18 each, independently, is hydrogen, C1-8 alkyl or acetyl, (xi) C1-8 alkyl, C1-8 alkoxy or C1-8 alkylthio substituted by Cyc1, or R1 and R2, taken together with carbon atoms which are attached to each of them, is 
in which Cyc1 is C3-15 mono-, bi- or tri-carbocyclic ring or 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, the above carbocyclic ring or heterocyclic ring may be substituted by one or more of (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) nitro, (iv) halogen atom, (v) nitrile, (vi) hydroxy, (vii) benzyloxy, (viii) xe2x80x94NR101R102, in which R101 and R102 each, independently, is hydrogen or C1-8 alkyl, (ix) xe2x80x94COOR103, in which R103 is hydrogen or C1-8 alkyl, (x) trihalomethyl, (xi) trihalomethoxy, (xii) phenyl, (xiii) phenyloxy, (xiv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, phenyloxy, hydroxy, xe2x80x94NR101R102 or xe2x80x94COOR103; 
is C3-7 mono-carbocyclic ring or 3-7 membered mono-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur;
R3 is
1) hydrogen,
2) C1-8 alkyl,
3) C2-8 alkenyl,
4) C1-8 alkoxy,
5) C1-8 alkylthio,
6) halogen atom,
7) nitro,
8) cyano,
9) hydroxy,
10) formyl,
11) C2-5 acyl,
12) xe2x80x94NR4R5, in which R4 and R5 each, independently, is hydrogen, C1-8 alkyl or acetyl,
13) xe2x80x94COOR6, in which R6 is hydrogen or C1-8 alkyl,
14) xe2x80x94CONR19R20, in which R19 and R20 each, independently, is hydrogen, C1-8 alkyl, phenyl, or C1-4 alkyl substituted by hydroxy, 5-7 membered mono-heterocyclic ring containing 1-2 of nitrogen(s), or 1 of nitrogen and 1 of oxygen, or R19 and R20, taken together is xe2x95x90CHxe2x80x94NR21R22, in which R21 and R22 each, independently, is hydrogen or C1-4 alkyl,
15) trihalomethyl,
16) trihalomethoxy,
17) phenyl,
18) phenyloxy,
19) phenylthio, or
20) C1-8 alkyl, C1-8 alkoxy, C1-8 alkylthio or C1-8 alkylamino substituted by phenyl, or
21) C1-8 alkyl or C2-8 alkenyl substituted by 1 or 2 of hydroxy, C1-4 alkoxy, phenoxy, halogen atom, nitrile, C2-5 acyl, xe2x80x94COOR6, xe2x80x94CONR19R20 or xe2x80x94NR4R5;
n is 0 or 1-5;
J is nitrogen atom or Cxe2x80x94R7;
R7 is
1) hydrogen,
2) C1-8 alkyl,
3) Cyc2,
4) C1-8 alkyl substituted by Cyc2,
5) C1-8 alkyl or C1-8 alkoxy substituted by 1-17 of halogen atom, or
6) halogen atom,
in which Cyc2 is C3-15 mono-, bi- or tri-carbocyclic ring or 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, the above carbocyclic ring or heterocyclic ring may be substituted by one or more of (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) nitro, (iv) halogen atom, (v) nitrile, (vi) hydroxy, (vii) benzyloxy, (viii) xe2x80x94NR201R202, in which R201 and R202 each, independently, is hydrogen or C1-8 alkyl, (ix) xe2x80x94COOR203, in which R203 is hydrogen or C1-8 alkyl, (x) trihalomethyl, (xi) trihalomethoxy, (xii) phenyl, (xiii) phenyloxy, (xiv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, phenyloxy, hydroxy, xe2x80x94NR201R202 or xe2x80x94COOR203;
E is a single bond, C1-4 alkylene, oxygen atom, sulfur atom, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, C1-4 alkylene-Mxe2x80x94, with the proviso that alkylene bond to ring and M is bond to G;
M is oxygen atom, sulfur atom, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94;
G is
1) C1-8 alkyl,
2) C2-8 alkenyl,
3) C2-8 alkynyl,
4) Cyc3, or
5) C1-8 alkyl substituted by xe2x80x94OR8, xe2x80x94SR8, xe2x80x94NR9R10, xe2x80x94COR11 or Cyc3, with the proviso that (i) one carbon atom in C1-8 alkyl, which is a component atom of cycloalkyl, may represent 3-7 membered cycloalkyl, or (ii) neighboring two carbon atom in C1-8 alkyl, which are component atoms of cycloalkyl, may represent 3-7 membered cycloalkyl;
in which Cyc3 is C3-15 mono-, bi- or tri-carbocyclic ring or 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, the above carbocyclic ring or heterocyclic ring may be substituted by one or more of (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) nitro, (iv) halogen atom, (v) nitrile, (vi) hydroxy, (vii) benzyloxy, (viii) xe2x80x94NR301R302, in which R301 and R302 each, independently, is hydrogen or C1-8 alkyl, (ix) xe2x80x94COOR303, in which R303 is hydrogen or C1-8 alkyl, (x) trihalomethyl, (xi) trihalomethoxy, (xii) phenyl, (xiii) phenyloxy, (xiv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, phenyloxy, hydroxy, xe2x80x94NR301R302 or xe2x80x94COOR303;
R8 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C1-8 alkyl substituted by phenyl or C1-8 alkoxy, or xe2x80x94Sxe2x80x94(C1-8 alkylene)-OR23, in which R23 is hydrogen or C1-8 alkyl; with the proviso that (i) one carbon atom in C1-8 alkylene, which is a component atom of cycloalkyl, may represent 3-7 membered cycloalkyl , or (ii) neighboring two carbon atom in C1-8 alkylene, which are component atoms of cycloalkyl, may represent 3-7 membered cycloalkyl;
R9 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C1-8 alkyl substituted by phenyl or C1-8 alkoxy;
R10 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C1-8 alkyl substituted by phenyl, or C2-5 acyl;
R11 is (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) hydroxy, (iv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, or (v) xe2x80x94NR12R13, in which R12 and R13 each, independently, is hydrogen, C1-8 alkyl or C1-8 alkyl substituted by phenyl;  is a single bond or a double bond;
with the proviso that the compounds in which R2 is C1-8 alkyl, E is a single bond or C1-4 alkylene and G is C1-8 alkyl are excluded;
or non-toxic acid thereof as active ingredient,
(ii) novel fused pyrazine derivatives of the formula (I): 
wherein R1 and R2each, independently, is (i) hydrogen, (ii) C1-8 alkyl, (iii) C1-8 alkoxy, (iv) C1-8 alkylthio, (v) Cyc1, (vi) nitrile, (vii) formyl, (viii) xe2x80x94COOR14, in which R14 is hydrogen or C1-8 alkyl, (ix) xe2x80x94CONR15R16, in which R15 and R16 each, independently, is hydrogen, C1-8 alkyl or phenyl, (x) C1-8 alkyl or C2-8 alkenyl substituted by 1 or 2 of hydroxy, C1-4 alkoxy, phenoxy, halogen atom, nitrile, C2-5 acyl, xe2x80x94COOR14, xe2x80x94CONR15R16, or xe2x80x94NR17R18, in which R17 and R18 each, independently, is hydrogen, C1-8 alkyl or acetyl, (xi) C1-8 alkyl, C1-8 alkoxy or C1-8 alkylthio substituted by Cyc1, or R1 and R2, taken together with carbon atoms which are attached to each of them, is 
in which Cyc1 is C3-15 mono-, bi- or tri-carbocyclic ring or 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, the above carbocyclic ring or heterocyclic ring may be substituted by one or more of (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) nitro, (iv) halogen atom, (v) nitrile, (vi) hydroxy, (vii) benzyloxy, (viii) xe2x80x94NR101R102, in which R101 and R102 each, independently, is hydrogen or C1-8 alkyl, (ix) xe2x80x94COOR103, in which R103 is hydrogen or C1-8 alkyl, (x) trihalomethyl, (xi) trihalomethoxy, (xii) phenyl, (xiii) phenyloxy, (xiv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, phenyloxy, hydroxy, xe2x80x94NR101R102 or xe2x80x94COOR103; 
is C3-7 mono-carbocyclic ring or 3-7 membered mono-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur;
R1 is
1) hydrogen,
2) C1-8 alkyl,
3) C2-8 alkenyl,
4) C1-8 alkoxy,
5) C1-8 alkylthio,
6) halogen atom,
7) nitro,
8) cyano,
9) hydroxy,
10) formyl,
11) C2-5 acyl,
12) xe2x80x94NR4R5, in which R4 and R5 each, independently, is hydrogen, C1-8 alkyl or acetyl,
13) xe2x80x94COOR6, in which R6 is hydrogen or C1-8 alkyl,
14) xe2x80x94CONR19R20, in which R19 and R20 each, independently, is hydrogen, C1-8 alkyl, phenyl, or C1-4 alkyl substituted by hydroxy, 5-7 membered mono-heterocyclic ring containing 1-2 of nitrogen(s), or 1 of nitrogen and 1 of oxygen, or R19 and R20, taken together is xe2x95x90CHxe2x80x94NR21R22, in which R21 and R22 each, independently, is hydrogen or C1-4 alkyl,
15) trihalomethyl,
16) trihalomethoxy,
17) phenyl,
18) phenyloxy,
19) phenylthio, or
20) C1-8 alkyl, C1-8 alkoxy, C1-8 alkylthio or C1-8 alkylamino substituted by phenyl, or
21) C1-8 alkyl or C2-8 alkenyl substituted by 1 or 2 of hydroxy, C1-4 alkoxy, phenoxy, halogen atom, nitrile, C2-5 acyl, xe2x80x94COOR6, xe2x80x94CONR19R20 or xe2x80x94NR4R5;
n is 0 or 1-5;
J is nitrogen atom or Cxe2x80x94R7;
R7 is
1) hydrogen,
2) C1-8 alkyl,
3) Cyc2,
4) C1-8 alkyl substituted by Cyc2,
5) C1-8 alkyl or C1-8 alkoxy substituted by 1-17 of halogen atom, or
6) halogen atom,
in which Cyc2 is C3-15 mono-, bi- or tri-carbocyclic ring or 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, the above carbocyclic ring or heterocyclic ring may be substituted by one or more of (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) nitro, (iv) halogen atom, (v) nitrile, (vi) hydroxy, (vii) benzyloxy, (viii) xe2x80x94NR201R202, in which R201 and R202 each, independently, is hydrogen or C1-8 alkyl, (ix) xe2x80x94COOR203, in which R203 is hydrogen or C1-8 alkyl, (x) trihalomethyl, (xi) trihalomethoxy, (xii) phenyl, (xiii) phenyloxy, (xiv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, phenyloxy, hydroxy, xe2x80x94NR201R202 or xe2x80x94COOR203;
E is a single bond, C1-4 alkylene, oxygen atom, sulfur atom, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, C1-4 alkylene-Mxe2x80x94, with the proviso that alkylene bond to ring and M is bond to G;
M is oxygen atom, sulfur atom, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94;
G is
1) C1-8 alkyl,
2) C2-8 alkenyl,
3) C2-8 alkynyl,
4) Cyc3, or
5) C1-8 alkyl substituted by xe2x80x94OR8, xe2x80x94SR8, xe2x80x94NR9R10, xe2x80x94COR11 or Cyc3, with the proviso that (i) one carbon atom in C1-8 alkyl, which is a component atom of cycloalkyl, may represent 3-7 membered cycloalkyl, or (ii) neighboring two carbon atom in C1-8 alkyl, which are component atoms of cycloalkyl, may represent 3-7 membered cycloalkyl;
in which Cyc3 is C3-15 mono-, bi- or tri-carbocyclic ring or 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, the above carbocyclic ring or heterocyclic ring may be substituted by one or more of (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) nitro, (iv) halogen atom, (v) nitrile, (vi) hydroxy, (vii) benzyloxy, (viii) xe2x80x94NR301R302, in which R301 and R302 each, independently, is hydrogen or C1-8 alkyl, (ix) xe2x80x94COOR303, in which R303 is hydrogen or C1-8 alkyl, (x) trihalomethyl, (xi) trihalomethoxy, (xii) phenyl, (xiii) phenyloxy, (xiv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, phenyloxy, hydroxy, xe2x80x94NR301R302 or xe2x80x94COOR303;
R8 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C1-8 alkyl substituted by phenyl or C1-8 alkoxy, or xe2x80x94Sxe2x80x94(C1-8 alkylene)-OR23, in which R23 is hydrogen or C1-8 alkyl; with the proviso that (i) one carbon atom in C1-8 alkylene, which is a component atom of cycloalkyl, may represent 3-7 membered cycloalkyl, or (ii) neighboring two carbon atom in C1-8 alkylene, which are component atoms of cycloalkyl, may represent 3-7 membered cycloalkyl;
R9 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C1-8 alkyl substituted by phenyl or C1-8 alkoxy;
R10 is hydrogen, C1-8 alkyl, C2-8 alkenyl, C1-8 alkyl substituted by phenyl, or C2-5 acyl;
R11 is (i) C1-8 alkyl, (ii) C1-8 alkoxy, (iii) hydroxy, (iv) C1-8 alkyl or C1-8 alkoxy substituted by phenyl, or (v) xe2x80x94NR12R13, in which R12 and R13 each, independently, is hydrogen, C1-8 alkyl or C1-8 alkyl substituted by phenyl;  is a single bond or a double bond;
with the proviso that the compounds in which R2 is C1-8 alkyl, E is a single bond or C1-4 alkylene and G is C1-8 alkyl and the following compounds of (1)-(14) are excluded;
(1) 4-(4-Chlorophenyl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline,
(2) 4-(Pyrimidine-2-yl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline,
(3) 4-Methoxycarbonylmethylthio(5-methyl-1,2,4-triazolo)-[4,3-a]quinoxaline,
(4) 4-Phenylthio-8-chloro(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline,
(5) 4-Phenylmethylthio(1,2,4-triazolo)-[4,3-a]quinoxaline,
(6) 4-(2-Chlorophenyl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline,
(7) 4-(4-Methoxyphenyl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline,
(8) 4-Allylthio(1,2,4- triazolo)-[4,3-a]quinoxaline,
(9) 4-(4-Chlorophenyl)thio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline,
(10) 4-Phenylmethylthio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline,
(11) 4-(Pyridin-2-yl)thio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline,
(12) 4-Phenylthio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline,
(13) 4-(4-Methoxyphenyl)thio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline, and
(14) 4-Phenyl(5-methyl-1,2,4-triazolo)-[4,3-a]quinoxaline;
or non-toxic salts thereof, and
(iii) a process for the preparation of fused pyrazine derivatives of the formula (I) and non-toxic salts thereof.
Unless otherwise specified, all isomers are included in the present invention. For example, alkyl, alkoxy and alkylene include straight and branched isomers. Double bond in alkenylene includes structure of configurations E, Z and EZ mixture. Isomers resulting from the presence of asymmetric carbon(s) e.g. branched alkyl, alkoxy and alkylene are also included within the present invention.
In the present invention, C1-4 alkyl is methyl, ethyl, propyl, butyl and isomeric groups thereof.
C1-8 alkyl is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl and isomeric groups thereof.
C1-8 alkoxy is methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy and isomeric groups thereof.
C1-8 alkylthio is methylthio, ethylthio, propylthio, butylthio, pentylthio, hexylthio, heptylthio, octylthio and isomeric groups thereof.
Halogen atom is chlorine, bromine, fluorine, or iodine.
Trihalomethyl is methyl tri-substituted by chlorine, bromine, fluorine, or iodine.
Trihalomethoxy is methoxy tri-substituted by chlorine, bromine, fluorine, or iodine.
C1-8 alkylamino is methylamino, ethylamino, propylamino, butylamino, pentylamino, hexylamino, heptylamino, octylamino and isomeric groups thereof.
C1-8 alkyl substituted by 1-17 of halogen atom is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl and isomeric groups thereof substituted by 1-17 of chlorine, bromine, fluorine, or iodine.
C1-8 alkoxy substituted by 1-17 of halogen atom is methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy and isomeric groups thereof substituted by 1-17 of chlorine, bromine, fluorine, or iodine.
C1-4 alkylene is methylene, ethylene, trimethylene, tetramethylene and isomeric groups thereof.
C2-8 alkenyl is vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, butadienyl, pentadienyl, hexadienyl, heptadienyl, octadienyl, hexatrienyl, heptatrienyl, octatrienyl and isomeric groups thereof.
C2-8 alkynyl is ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl and isomeric groups thereof.
C2-5 acyl is acetyl, propionyl, butyryl, pentanoyl and isomeric groups thereof.
C3-7 mono-carbocyclic ring is, for example, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, benzene.
3-7 membered mono-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur is 3-7 membered mono-heterocyclic aryl containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, partially or fully saturated thereof.
3-7 membered mono-heterocyclic aryl containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and 1 or 1 of sulfur is, for example, pyrrole, imidazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, azepine, diazepine, furan, pyran, oxepin, oxazepine, thiophene, thiain (thiopyran), thiepin, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, oxazine, oxadiazine, oxazepine, oxadiazepine, thiadiazole, thiazine, thiadiazine, thiazepine, thiadiazepine.
Partially or fully saturated 3-7 membered mono-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur is, for example, oxirane, aziridine, azetidine, pyrroline, pyrrolidine, imidazoline, imidazolidine, triazoline, triazolidine, tetrazoline, tetrazolidine, pyrazoline, pyrazolidine, piperidine, piperazine, tetrahydropyrimidine, tetrahydropyridazine, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyran, dihydrothiophene, tetrahydrothiophene, dihydrothiain (dihydrothiopyran), tetrahydrothiain (tetrahydrothiopyran), dihydrooxazole, tetrahydrooxazale, dihydroisoxazole, tetrahydroisoxazole, dihydrothiazole, tetrahydrothiazole, dihydroisothiazole, tetrahydroisothiazole, morpholine, thiomorpholine, oxolane, oxane.
C3-15 mono-, bi- or tri-carbocyclic ring is, for example, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, benzene, pentalene, indene, naphthalene, azulene, fluorene, phenanthrene, anthracene, acenaphthylene, biphenylene, perhydropentalene, perhydroindene, perhydronaphthalene, perhydroazulene, perhydrofluorene, perhydrophenanthrene, perhydroanthracene, perhydroacenaphthylene, perhydrobiphenylene.
5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur is 5-18 membered mono-, bi- or tri-heterocyclic aryl containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, partially or fully saturated thereof.
5-18 membered mono-, bi- or tri-heterocyclic aryl containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, is, for example, pyrrole, imidazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, azepine, diazepine, furan, pyran, oxepin, oxazepine, thiophene, thiain (thiopyran), thiepin, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, oxazine, oxadiazine, oxazepine, oxadiazepine, thiadiazole, thiazine, thiadiazine, thiazepine, thiadiazepine, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, isobenzothiophene, indazole, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, benzoxazole, benzothiazole, benzoimidazole, carbazole or acridine.
Partially or fully saturated 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1-2 of oxygen(s) and/or 1 of sulfur, for example, pyrroline, pyrrolidine, imidazoline, imidazolidine, triazoline, triazolidine, tetrazoline, tetrazolidine, pyrazoline, pyrazolidine, piperidine, piperazine, tetrahydropyrimidine, tetrahydropyridazine, dihydrofuran, tetrahydrofuran, dihydropyran, tetrahydropyran, dihydrothiophene, tetrahydrothiophene, dihydrothiain (dihydrothiopyran), tetrahydrothiain (tetrahydrothiopyran), dihydrooxazole, tetrahydrooxazole, dihydroisoxazole, tetrahydroisoxazole, dihydrothiazole, tetrahydrothiazole, dihydroisothiazole, tetrahydroisothiazole, morpholine, thiomorpholine, oxolane, oxane, indoline, isoindoline, dihydrobenzofuran, perhydrobenzofuran, dihydroisobenzofuran, perhydroisobenzofuran, dihydrobenzothiophene, perhydrobenzothiophene, dihydroisobenzothiophene, perhydroisobenzothiophene, dihydroindazole, perhydroindazole, dihydroquinoline, tetrahydroquinoline, perhydroquinoline, dihydroisoquinoline, tetrahydroisoquinoline, perhydroisoquinoline, dihydrophthalazine, tetrahydrophthalazine, perhydrophthalazine, dihydronaphthyridine, tetrahydronaphthyridine, perhydronaphthyridine, dihydroquinoxaline, tetrahydroquinoxaline, perhydroquinoxaline, dihydroquinazoline, tetrahydroquinazoline, perhydroquinazoline, dihydrocinnoline, tetrahydrocinnoline, perhydrocinnoline, dihydrobenzoxazole, perhydrobenzoxazole, dihydrobenzothiazole, perhydrobenzothiazole, dihydrobenzimidazole, perhydrobenzimidazole, benzoxazepine, benzoxadiazepine, benzothiazepine, benzothiadiazepine, benzoazepine, benzodiazepine, indolooxazepine, indolotetrahydrooxazepine, indolooxadiazepine, indolotetrahydrooxadiazepine, indolothiazepine, indolotetrahydrothiazepine, indolothiadiazepine, indolotetrahydrothiadiazepine, indoloazepine, indolotetrahydroazepine, indolodiazepine, indolotetrahydrodiazepine, benzofurazan, benzothiadiazole, benzotriazole, camphor, imidazothiazole, dihydrocarbazole, tetrahydrocarbazole, perhydrocarbazole, dihydroacridine, tetrahydroacridine, perhydroacridine.
5-7 membered mono-heterocyclic ring containing 1-2 of nitrogen(s), or 1 of nitrogen and 1 of oxygen is, for example, pyrrole, pyrroline, pyrrolidine, imidazole, pyrazole, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyridine, pyridazine, pyrimidine, pyrazine, piperidine, piperazine, oxazole, isoxazole, oxazine, morpholine.
C3-7 cycloalkyl represented by one carbon atom in C1-8 alkyl or C1-8 alkylene, which is a component atom of cycloalkyl, is, for example, in case of C2 alkyl or alkylene, 
in case of C3 alkyl or alkylene, 
C3-7 cycloalkyl represented by neighboring two carbon atoms in C1-8 alkyl or C1-8 alkylene, which is component atoms of cycloalkyl, is , for example, in case of C4 alkyl or alkylene, 
Salts
Non-toxic salts of the present invention include all pharmaceutically acceptable salts, for example, general salts, acid addition salts, hydrate salts.
The compounds of formulae (I) of the present invention may be converted into the corresponding salts. Non-toxic salts and water-soluble salts are preferred. Suitable salts, for example, include: salts of alkali metals (e.g. potassium, sodium), salts of alkaline earth metals (e.g. calcium, magnesium), ammonium salts, salts of pharmaceutically acceptable organic amines (e.g. tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)amine, lysine, arginine, N-methyl-D-glucamine).
The compounds of formulae (I) may be converted into the corresponding acid addition salts. Non-toxic acid addition salts and water-soluble acid addition salts are preferred. Suitable salts, for example, include: salts of inorganic acids e.g. hydrochloride, hydrobromide, sulfate, phosphate, nitrate; salts of organic acids e.g. acetate, trifluoroacetate, lactate, tartarate, oxalate, fumarate, maleate, citrate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, toluenesulphonate, isethionate, glucuronate, gluconate.
The compounds of formulae (I) and salts thereof may be converted into the corresponding hydrates by conventional means.
In the compounds of the present invention of formulae (1), the following compounds of the formulae are preferred:
the formula (I-A): 
wherein all the symbols are as hereinbefore defined;
the formula (I-B): 
wherein all the symbols are as hereinbefore defined;
the formula (I-C): 
wherein all the symbols are as hereinbefore defined;
the formula (I-D): 
wherein all the symbols are as hereinbefore defined;
the formula (I-E): 
wherein all the symbols are as hereinbefore defined;
the formula (I-F): 
wherein all the symbols are as hereinbefore defined;
the formula (I-G): 
wherein R1G and R2G each, independently, is (i) hydrogen, (ii) C1-8 alkyl, (iii) C1-8 alkoxy, (iv) C1-8 alkylthio, (v) Cyc1, (vi) nitrile, (vii) formyl, (viii) xe2x80x94COOR14, (ix) xe2x80x94CONR15R16, (x) C1-8 alkyl or C2-8 alkenyl substituted by 1 or 2 of hydroxy, C1-4 alkoxy, phenoxy, halogen atom, nitrile, C2-5 acyl, xe2x80x94COOR14, xe2x80x94CONR15R16, or xe2x80x94NR17R18, (xi) C1-8 alkyl, C1-8 alkoxy or C1-8 alkylthio substituted by Cyc1, and the other symbols are as hereinbefore defined;
the formula (I-H): 
wherein GH is substituted or unsubstituted C3-15 mono-, bi- or tri-carbocyclic ring, and the other symbols are as hereinbefore defined;
the formula (I-J): 
wherein GJ is C1-8 alkyl, and the other symbols are as hereinbefore defined;
the formula (I-K): 
wherein GK is C1-8 alkyl substituted by hydroxy, and the other symbols are as hereinbefore defined;
the formula (I-L): 
wherein GL is substituted or unsubstituted 5-18 membered mono-, bi- or tri-heterocyclic ring containing 1-4 of nitrogen(s), 1 of oxygen and/or 1 of sulfur, and the other symbols are as hereinbefore defined.
The following compounds, the compounds described in Table 1-Table 15 and the compounds described in the Examples or non-toxic salts thereof are more preferred.
The following known compounds of (1)-(14) are on the market, but they are not known as adhesion molecules expression inhibitors at all.
Compound (1):
4-(4-Chlorophenyl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 10E-957) 
Compound (2):
4-(Pyrimidine-2-yl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 11E-909) 
Compound (3):
4-Methoxycarbonylmethylthio(5-methyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 12E-941) 
Compound (4):
4-Phenylthio-8-chloro(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 12E-954) 
Compound (5):
4-Phenylmethylthio(1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-912) 
Compound (6):
4-(2-Chlorophenyl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-913) 
Compound (7):
4-(4-Methoxyphenyl)thio(1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-914) 
Compound (8):
4-Allylthio(1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-922) 
Compound (9):
4-(4-Chlorophenyl)thio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet a catalog No. 4G-937) 
Compound (10):
4-Phenylmethylthio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-938) 
Compound (11):
4-(Pyridin-2-yl)thio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-943) 
Compound (12):
4-Phenylthio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-944) 
Compound (13):
4-(4-Methoxyphenyl)thio(5-trifluoromethyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Bionet; a catalog No. 4G-945) 
Compound (14):
4-Phenyl(5-methyl-1,2,4-triazolo)-[4,3-a]quinoxaline (Labotest; a catalog No. LT-2 VO 14) 
Process for the Preparation
The compounds of the present invention of the formula (I), may be prepared by following methods or the methods described in the Examples.
[1] In the compounds of the present invention of the formula (I), the compound in which R3 is not xe2x80x94NH2, that is the compound of the formula (I-1): 
wherein R1-1 and R2-1 each, independently, is the a same meaning as R1 and R2, with the proviso that, R3 in R1-1 and R2-1 is not xe2x80x94NH2, the other symbols are as hereinbefore defined;
may be prepared by following methods (a)-(d).
(a) The compound in which E is oxygen atom, sulfur atom, xe2x80x94(C1-4 alkylene)-Oxe2x80x94 or xe2x80x94(C1-4 alkylene)-Sxe2x80x94, that is the compound of the formula (I-1-a): 
wherein Ea is oxygen atom, sulfur atom, xe2x80x94(C1-4 alkylene)-Oxe2x80x94 or xe2x80x94(C1-4 alkylene)-Sxe2x80x94, and the other symbols are as hereinbefore defined;
may be prepared by reacting the compound of the formula (II-a-1): 
wherein X is an ordinary elimination group (e.g. chloride, bromide, iodide, mesyl or tosyl), and the other symbols are as hereinbefore defined;
or the compound of the formula (II-a-2): 
wherein L is C1-4 alkylene, and the other symbols are as hereinbefore defined;
with the compound of the formula (III-a):
Hxe2x80x94Eaxe2x80x94Gxe2x80x83xe2x80x83(III-a)
wherein all the symbols are as hereinbefore defined.
The reaction of the compound of the formula (II-a-1) or the formula (II-a-2), and the compound of the formula (III-a) was known, for example, it may be carried out by reacting alcohol or thiol of the formula (III-a) in an inactive organic solvent (e.g. dimethylformamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran or acetone), using an alkali metal hydride, an alkali metal hydroxide, (e.g. sodium hydroxide, potassium hydroxide or lithium hydroxide), an alkaline earth metal hydroxide (e.g. barium hydroxide or calcium hydroxide) or a carbonate (e.g. sodium carbonate or potassium carbonate), an aqueous solution thereof or mixture thereof at 0-40xc2x0 C., and reacting the obtained alkoxide ion or thiolate ion and the compound of the formula (II-a-1) or the formula (II-a-2) at 0-40xc2x0 C.
This reaction may be carried out under an inert gas (e.g. argon, nitrogen) to avoid water in order to obtain a preferable result.
(b) The compound in which J is Cxe2x80x94R7 and E is a single bond or C1-4 alkylene, that is the compound of the formula (I1-b): 
wherein Eb is a single bond or C1-4 alkylene, the other symbols are as hereinbefore defined;
may be prepared by reacting the compound (II-b): 
wherein all the symbols are as hereinbefore defined;
with the compound of the formula (III-b-1):
R7xe2x80x94COOHxe2x80x83xe2x80x83(III-b-1)
wherein all the symbols are as hereinbefore defined;
or the compound of the formula (III-b-2): 
wherein all the symbols are as hereinbefore defined;
or the compound of the formula (III-b-3):
R7xe2x80x94C(OT)3xe2x80x83xe2x80x83(III-b-3)
wherein T is C1-8 alkyl, the other symbols are as hereinbefore defined.
The reaction of the compound of the formula (II-b) and the compound of the formula (III-b-1), the formula (III-b-2) or the formula (III-b-3) was known (See J Med. Chem., 33, 2240 (1990), J. Heterocyclic Chem. 31, 549 (1994).), for example, it may be carried out in organic solvent (e.g. pyridine, toluene, benzene, dichloromethane, 1,2-dichloroethane, methanol, ethanol) or without a solvent at 20-150xc2x0 C.
(c) The compound in which J is nitrogen atom and E is a single bond or C1-4 alkylene, that is the compound of the formula (I-1-c): 
wherein all the symbols are as hereinbefore defined;
may be prepared by reacting the compound (II-c): 
wherein all the symbols are as hereinbefore defined;
with an alkali metal azide.
The reaction of the compound of the formula (II-c) and an alkali metal azide was known (See J. Med. Chem. 35, 3323 (1992).), for example, it may be carried out in an alcohol solvent (e.g. methanol, ethanol), in the presence of an inorganic acid (e.g. hydrochloric acid or sulfuric acid) using an alkali metal azide (e.g. sodium azide) at 40-120xc2x0 C.
(d) The compound in which E is xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94(C1-4 alkylene)-SOxe2x80x94 or xe2x80x94(C1-4 alkylene)-SO2xe2x80x94, that is the compound of the formula (I-1-d): 
wherein Ed is xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94(C1-4 alkylene)-SOxe2x80x94 or xe2x80x94(C1-4 alkylene)-SO2xe2x80x94, and the other symbols are as hereinbefore defined;
may be prepared by subjecting the compound in which E is sulfur atom or xe2x80x94(C1-4 alkylene)-Sxe2x80x94 in the above compound of the formula (I-1-a), that is the compound of the formula (I-1-a-1): 
wherein Ea-1 is sulfur atom or xe2x80x94(C1-4 alkylene)-Sxe2x80x94, and the other symbols are as hereinbefore defined;
to oxidation reaction.
The oxidation reaction was known, for example, in case of the compound in which Ed is xe2x80x94SOxe2x80x94 or xe2x80x94(C1-4 alkylene)-SOxe2x80x94, it may be carried out in a suitable organic solvent (e.g. dichloromethane, chloroform, benzene, hexane, t-butyl alcohol), in the presence of 1 equivalent oxidizing agent (e.g. hydrogen peroxide, sodium periodate, acyl nitrite, sodium perborate, peroxide (such as 3-chloroperbenzoic acid, peracetic acid)) at xe2x88x9278-0xc2x0 C.
In case of the compound in which Ed is xe2x80x94SO2xe2x80x94 or xe2x80x94(C1-4 alkylene)-SO2xe2x80x94, it may be carried out in a suitable organic solvent (e.g. dichloromethane, chloroform, benzene, hexane, t-butyl alcohol), in the presence of an excess amount of oxidizing agent (e.g. hydrogen peroxide, sodium periodate, acyl nitrite, sodium perborate, peroxide (such as 3-chloroperbenzoic acid, peracetic acid)) at 0-40xc2x0 C.
[2] In the compounds of the present invention of the formula (I), the compound in which R3 is xe2x80x94NH2, that is the compound of the formula (I2): 
wherein R1-2 and R2-2 each, independently, is the a same meaning as R1 and R2, with the proviso that, R3 in R1-2 and R2-2 is xe2x80x94NH2, the other symbols are as hereinbefore defined;
may be prepared by subjecting the compound in which R3 is nitro in the compound of formula (I-1), that is the compound of the formula (I-1-2): 
wherein R1-1-2 and R2-1-2 each, independently, is the a same meaning as R1 and R2, with the proviso that, R3 in R1-1-2 and R2-1-2 is nitro, the other symbols are as hereinbefore defined;
to reduction.
The reduction of nitro was known, for example, it may be carried out by hydrogenolysis and reduction using an organic metal.
Hydrogenolysis was known, for example, it may be carried out in an inactive solvent [e.g. ether (such as tetrahydrofuran, dioxane, dimethoxyethane or diethyl ether), alcohol (such as methanol or ethanol), benzene (such as benzene or toluene), ketone (such as acetone or methyl ethyl ketone), nitrile (such as acetonitrile), amide (such as dimethylformamide), water, ethyl acetate, acetic acid or two more mixture thereof], in the presence of a catalyst (e.g. palladium on carbon, palladium black, palladium, palladium hydroxide, platinum dioxide, nickel or Raney-nickel), optionally in the presence of an inorganic acid (e.g. hydrochloric acid, sulfuric acid, hypochlorous acid, boric acid or tetrafluoroboric acid) or an organic acid (e.g. acetic acid, p-toluenesulfonic acid, oxalic acid, trifluoroacetic acid or formic acid), at ordinary or elevated pressure of hydrogen gas or in the presence of ammonium formate at 0-200xc2x0 C. It does not matter using a salt of acid, when it is carried out in the presence of an acid.
The reduction using an organic metal was known, for example, it may be carried out in a water miscible solvent (such as ethanol or methanol), optionally in the presence of an aqueous solution of hydrochloric acid using an organic metal (such as zinc, iron, tin, tin chloride, iron chloride) at 0-150xc2x0 C.
[3] In the compounds of the present invention of the formula (I), the compound in which R3 is xe2x80x94NR4-3R5-3, in which R4-3 and R5-3 each, independently, is the a same meaning as R4 and R5, with the proviso that, they are not hydrogen at same time, that is the compound of the formula (I3): 
wherein R1-3 and R2-3 each, independently, is the a same meaning as R1 and R2, with the proviso that, R3 in R1-3 and R2-3 is xe2x80x94NR4-3R5-3, and the other symbols are as hereinbefore defined;
may be prepared by subjecting the above compound of the formula (I-2) to alkylation or acetylation also.
The reaction of alkylation was known, for example, it may be carried out in a solvent (such as tetrahydrofuran dimethylformamide or a mixture thereof) using alkyl iodide (such as methyl iodide), in the presence of sodium hydride at 0-40xc2x0 C.
The reaction of acetylation was known, for example, it may be carried out in the presence of tertiary amine or pyridine, using anhydrous acetic acid at 0-80xc2x0 C.
[4] The compound in which R1 is (i) COOH, (ii) CONR15R16, (iii) formyl, (iv) nitrile, (v) C2-8 alkenyl, (vi) methyl substituted by halogen atom, hydroxy or phenoxy, (vii) ethenyl substituted by COOR14a, in which R14a is C1-8 alkyl; nitrile, halogen atom, acetyl, C1-6 alkyl or C2-6 alkenyl, and R2 is hydrogen, may be prepared by following methods described in scheme 1(1) and scheme 1(2) also.
In the schemes, X is halogen atom, Ph is phenyl, s is 0 or 1-6, Rx is hydrogen, halogen atom, C1-6 alkyl, C2-6 alkenyl, COOR14a, nitrile or acetyl, RY is hydrogen, C1-6 alkyl, halogen atom or nitrile, and the other symbols are as hereinbefore defined.
All reactions in scheme were known. 
[5] The compound in which n is 1, R3 is (i) formyl, (ii) C1-8 alkanoyl, (iii) C2-8 alkenyl, (iv) methyl substituted by halogen atom or xe2x80x94NR4R5, (v) C1-8 alkyl substituted by hydroxy, (vi) C2-8 alkenyl substituted by COOR6, hydroxy, halogen atom, nitrile, may be prepared by following methods described in scheme 2(1) and scheme 2(2) also.
In the schemes, t is 0 or 1-6, RW is hydrogen, C1-6 alkyl, C2-6 alkenyl, halogen atom, nitrile, COOR6a, in which R6a is C1-8 alkyl; RZ is hydrogen, C1-6 alkyl, halogen atom or nitrile, and the other symbols are as hereinbefore defined.
All reactions in scheme were known. 
The compounds of the formulae (II-a), (III-a), (II-b), (III-b-1), (III-b-2) are known per se or may be prepared by known methods (See J. Med. Chem. 33, 2240 (1990) or J. Med. Chem. 35, 3323 (1992).).
In each reaction in the present specification, products may be purified by conventional techniques. For example, purification may be carried out by distillation at atmospheric or reduced pressure, by high performance liquid chromatography, by thin layer chromatography or by column chromatography using silica gel or magnesium silicate, by washing or by recrystallization. Purification may be carried out after each reactions or after a series of reactions.
Pharmacological Activities
The inhibitory activity of adhesion molecules expression of the compound of the formula (I) was confirmed as below.
(1) Inhibition of Adhesion Molecules Expression in HUVEC
Human umbilical vein endothelial cells were cultured using MCDB104 medium (Nissui) containing 5% Fetal bovine serum supplemented antibiotics (100 U/ml penicillin, 100 xcexcg/ml streptomycin (Gibco)), 0.01 mg/ml heparin (Nissui) and 0.005 mg/ml endothelial cells (EC) growth factor (Nissui) in gelatin coated 96-well microtiter plates by resulting in confluent monolayers.
The compounds were dissolved in dimethyl sulfoxide and prepared final 0.2% concentration in the medium and then added 50 xcexcl into the well. Cytokine stimulation of EC was performed by adding 50 xcexcl of 10 ng/ml TNFxcex1 (Genzyme) (for E-selectin. and ICAM-1 expression) or 50 xcexcl of 10 ng/ml TNFxcex1 plus 1 ng/ml IL-4 (Genzyme)(for VCAM- expression).
After stimulation for 6 hours, the wells were washed with PBS(xe2x88x92) once and EC monolayers were fixed by incubating with methanol containing 0.3% hydrogen peroxide for 10 minutes at room temperature. After washing with PBS(xe2x88x92) three times, an ELISA was used to measure the expression of adhesion molecules on EC surface. The ELISA was performed at room temperature with three times washes with PBS(xe2x88x92) between each step. EC were incubated for 30 minutes in turn with 100 xcexcl primary antibody which were anti-ICAM-1 antibody (BBA4), anti-E-selectin antibody (BBA2) or anti-VCAM-1 antibody (BBA6) (mouse IgG1, 1.25 xcexcl/ml; RandD), with 100 xcexcl of secondary antibody which was goat anti-mouse IgG conjugated peroxidase (dilution (1/400); Nordic Immunological laboratories). The enzyme substrate 1 mg/ml o-phenylenediamine/2HCl and 0.1% hydrogen peroxide of 100 xcexcl was added and incubated for 4 minutes at room temperature.
Reaction was stopped with 50 xcexcl of 8N sulfuric acid and optical density (OD) of each well read at 490 nm in immunoreder. Inhibitory percentage was calculated as follows and IC50 value was estimated by percentage for control.
Inhibitory percentage (%)=[(Cxe2x88x92S)/(Cxe2x88x92B)]xc3x97100
C: OD of the sample stimulated by cytokines
S: OD of the sample stimulated by cytokines in the presence of test compound
B: OD of the nonstimulated sample
These results are shown in Table 16.
Furthermore, MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay was enforced and the number of living cells was counted in order to estimate the effect of compound not to depend cytotoxicity on the above evaluation system.
(2) Cytotoxicity Test by MTT Assay
[Method]
EC Were treated under conditions identical to those employed for measurement of adhesion molecules expression. After incubation, the wells were washed with PBS(xe2x88x92) once, added 100 xcexcl of MTT solution (1 mg/ml) and incubated for 3 hours. After incubation supernatants were discarded and 100 xcexcl of methanol added. After stirring, OD of each well read at 570 nm/690 nm in immunoreader. Inhibitory percentage was calculated as follows and IC50 value was estimated by percentage for control.
Inhibitory percentage (%)=[(Cxe2x88x92S)/C)]xc3x97100
C: OD of the sample stimulated by cytokines
S: OD of the sample stimulated by cytokines in the presence of test compound
As the result, IC50 values of compound (12), Example 3 (16) and Example 3 (73) were more than 50 xcexcM, 100 xcexcM and 25 xcexcM respectively.
Toxicity
The toxicity of the compounds of the present invention is very low and therefore, the compounds may be considered safe for pharmaceutical use.
Application for Pharmaceuticals
Inhibition of adhesion molecules expression is useful for prevention and or treatment of diseases, for example, inflammation, rheumatoid arthritis, allergies, asthma, atopic dermatitis, psoriasis, suppression of ischemia reperfusion injury, nephritis, hepatitis, multiple sclerosis, ulcerative colitis, ARDS, suppression of transplant rejection, sepsis, diabetes, autoimmune diseases, tumor metastasis, arteriosclerosis and AIDS in animals including human beings, especially human beings.
For the purpose above described, the compounds of formulae (I) of the present invention, non-toxic salts, acid addition salt or hydrates thereof may be normally by administered systematically or locally, usually by oral or parenteral administration.
The doses to be administered are determined depending upon, for example, age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment In the human adult, the doses per person are Generally from 1 mg to 1000 mg, by oral administration, up to several times per day, and from 1 mg to 100 mg, by parenteral administration (preferably intravenous administration), up to several times per day, or continuous administration from 1 to 24 hours per day from vein.
As mentioned above, the doses to be used depend upon various conditions. Therefore, there are cases in which doses lower than or greater than the ranges specified above may be used.
The compounds of the present invention may be administered in the form of, for example, solid forms for oral administration, liquid forms for oral administration, injections, liniments or suppositories for parenteral administration.
Solid forms for oral administration include compressed tablets, pills, capsules, dispersible powders, and granules. Capsules include hard capsules and soft capsules.
In such solid forms, one or more of the active compound(s) may be admixed with vehicles (such as lactose, mannitol, glucose, microcrystalline cellulose, starch), binders (such as hydroxypropyl cellulose, polyvinylpyrrolidone or magnesium metasilicate aluminate), disintegrants (such as cellulose calcium glycolate), lubricants (such as magnesium stearate), stabilizing agents, and solution adjuvants (such as glutamic acid or aspartic acid) and prepared according to methods well known in normal pharmaceutical practice. The solid forms may, if desired, be coated with coating agents (such as sugar, gelatin, hydroxypropyl cellulose or hydroxypropylmethyl cellulose phthalate), or be coated with two or more films. And further, coating may include containment within capsules of absorbable materials such as gelatin.
Liquid forms for oral administration include pharmaceutically acceptable solutions, suspensions and emulsions, syrups and elixirs. In such forms, one or more of the active compound(s) may be dissolved, suspended or emulized into diluent(s) commonly used in the art (such as purified water, ethanol or a mixture thereof). Besides such liquid forms may also comprise some additives, such as wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, aroma, preservative or buffering agent.
Injections for parenteral administration include sterile aqueous, suspensions, emulsions and solid forms which are dissolved or suspended into solvent(s) for injection immediately before use. In injections, one or more of the active compound(s) may be dissolved, suspended or emulized into solvent(s). The solvents may include distilled water for injection, physiological salt solution, vegetable oil, propylene glycol, polyethylene glycol, alcohol, e.g. ethanol, or a mixture thereof.
Injections may comprise some additives, such as stabilizing agents, solution adjuvants (such as glutamic acid, aspartic acid or POLYSORBATE80 (registered trade mark)), suspending agents, emulsifying agents, soothing agent, buffering agents, preservative. They may be sterilized at a final step, or may be prepared and compensated according to sterile methods. They may also be manufactured in the form of sterile solid forms which may be dissolved in sterile water or some other sterile diluent(s) for injection immediately before use.
Other forms for parenteral administration include liquids for external use, ointments and endermic liniments, inhalations, sprays, suppositories and pessaries for vaginal administration which comprise one or more of the active compound(s) and may be prepared by methods known per se. Sprays may comprise additional substances other than diluents, such as stabilizing agents (such as sodium sulfate), isotonic buffers (such as sodium chloride, sodium citrate or citric acid). For preparation of such sprays, for example, the method described in the U.S. Pat. Nos. 2,868,691 or 3,095,355 may be used.
The Best Form in Order to Conduct a Present Invention
The following Reference Examples and Examples illustrate the present invention, but do not limit the present invention.
The solvents in the parentheses show the developing or eluting solvents and the ratios of the solvents used are by volume in chromatographic separations or TLC.
The solvents in the parentheses in NMR show the solvents used in measurement.